The expression of proteins heterologous to host cell has been accomplished with varying degree of success in a variety of host cells. For example, DNA encoding enzymes originating in the genome of one species of prokaryote have been integrated into plasmid and expressed and secreted from cells of a different prokaryotic species. Indeed, proteins originating in genera as varied as Homo sapiens, Bos, OVIS, Mus, Rattus, and others have been successfully expressed in, for example, E. coli, Saccharomyes, Streptomyces, Bacillus, and other recombinant host cells.
Notwithstanding the notable successes in, for example, the expression and secretion of insulin from E. coli as a fusion protein, the successful production and processing of proteins in association with secretion leaders is by no means a routinely achievable event. Numerous factors are entailed in the successful expression and secretion of a polypeptide in a recombinant host. The host may process certain codons encoding particular amino acids with greater fidelity than other codons encoding the same amino acid. The mRNA transcript may have the ability to form secondary structures with itself and such secondary structures may lead to decreased translation. The mRNA transcript may also form secondary structures with control sequences for the translation of the transcript such as the ribosome binding site, thereby interfering with the binding of the transcript to the ribosome and the efficient translation thereof.
After translation of the mRNA transcript has been successfully accomplished, the proper folding of the translated protein, as well as post-translational processing and secretion must also occur in order to obtain a biologically active recombinantly produced protein. Secretion, a proper folding of a protein and processing, which may be regarded as the cleavage of the mature protein from a secretion leader may be events that are separable or inseparable.
Pseudomonas exotoxin (PE) A is a powerful bacterial toxin that acts as an ADP-ribosyl transferase and inhibits protein synthesis in eukaryotic cells by catalyzing the transfer of the ADP ribosyl moiety from oxidized NAD onto elongation factor-2 (EF-2). The nucleotide sequence of PE and its cloning and expression in E. coli has been disclosed by Gray et al., Proc. Natl. Acad. Sci. USA 81: 2645-2649 (1984). Most importantly, Gray et al. report that the material so expressed is enzymatically active but is neither processed nor secreted. They suggest that all the components required for secretion of PE toxin from E. coli are not present in that host. Gray et al. also point out that PE and diphtheria toxin (DT) share certain characteristics including size, secretion as a single polypeptide chain having disulfide bridges and no free sulfhydryl groups, alteration in covalent structure from an inactive proenzyme to an enzymatically active state, similarity of site of enzymatic activity and maximal production in iron deficient medium. Despite these similarities, the two toxins are immunologically non-crossreactive, have different amino acid composition, differ in their mode of activation, and bind to different cell receptors. Furthermore, while intact DT binds ATP and possesses NAD glycohydrolase activity, PE lacks these properties. Lastly, computer analysis and DNA hybridization studies carried out by Gray et al. indicate no regions of significant homology between these proteins.
Mozola et al., J. Bacteriol., 159: 2, 683-687 (1984) report the cloning and expression in E. coli and Pseudomonas of fragments of the Pseudomonas exotoxin A DNA sequence derived from the chromosomal DNA of P. aeruginosa strain PA103. These cloned and expressed partial sequences, while having at least partial immunological identity with Pseudomonas exotoxin A by Western blot, were from the carboxyl terminal regions of the gene encoding the toxin. Further, supporting this conclusion by Mazola et al. that the NH.sub.2 -terminus of the gene was not present, was the observation that the protein was not secreted. The known published literature therefore indicates that secretion of Pseudomonas exotoxin A as a cloned product in both Pseudomonas and in other hosts is unprecedented.
Immunoconjugates of Pseudomonas exotoxin are known and methods for the making thereof are disclosed in U.S. Pat. No. 4,545,985.
It would be advantageous to produce Pseudomonas exotoxin A and other proteins in processed form in a convenient host. The production of processed proteins in which a leader sequence is removed from the mature protein would confer several advantages. First, such processed proteins would lack amino acids unnecessary in the mature form for the biological activity of the protein. If the processed protein is obtained and ultimately used parenterally in a patient, the absence of leader sequence amino acids would be expected to make the mature protein less immunogenic. In addition, processing of the leader sequence and production of the mature protein clearly accomplishes, in the recombinant host, a processing step that would otherwise be carried out by other chemical or enzymatic means. Processing of the leader sequence by the recombinant host and proper secretion may also, advantageously assist in the proper folding of the expressed protein product thereby to achieve biological activity without further in vitro processing steps.
Furthermore, processing by E. coli as the recombinant host to form a mature proteion, eliminates the necessity of placing an NH.sub.2 -terminal methionine at the NH.sub.2 -terminus of the mature protein since the start codon ATG is found at the signal or leader sequence NH.sub.2 -terminus. Such NH.sub.2 -terminal methionine residues may be immunogenic when formed as part of a mature protein and may be chemotactic.
In addition, such in vivo processing of the precursor protein by the recombinant host is involved in extracellular secretion of the processed protein. In most Gram-negative hosts, such secretion might be expected to be periplasmic. Suh periplasmic extracellular secretion can be expected to lead to savings in the processing to remove extraneous intracellular proteins produced by the recombinant host. Surprisingly, it has been found that at least with respect to Pseudomonas exotoxin A secretion in E. coli, is extramural, i.e., passes through the cell wall and accumulates in the growth medium.